The effects of nicotine on the transcriptional activity of the proENK gene, proenkephalin A (proENK) mRNA levels, and the secretion of [Met5]-enkephalin (ME) were studied in bovine adrenal medullary chromaffin (BAMC) cells. Nicotine (105M) caused an immediate secretion (within 1 hr) of ME followed by a delayed secretion (12-24 hr after treatment) into the medium. Post-treatment with the cholinergic antagonists up to 6 hr after the nicotine treatment signficantly inhibited the delayed secretion of ME induced by nicotine. However, nicotine-induced long-term secretion of ME was not affected when cholinergic antagonists were added 9 or 12 hr after the nicotine treatment. Long-term (24 hr) stimulation of BAMC cells with nicotine also increased proENK mRNA level. This nicotine-induced response was inhibited by post-treatment with cholinergic antagonists 0.5, 1,3, and 6 hr after the nicotine treatment. Like the secretion experiments, these cholinergic antagonists did not affect the nicotine-induced responses when they were added at 9 and 12 hr. Post-treatment with nimodipine (5M), calmidazolium (15M), or KN-62 (55M) up to 6 hr after the nicotine treatment significantly inhibited the increases of the long-term secretion of ME and proENK mRNA level induced by nicotine. However, these agents were ineffective in blocking the long-term secretion of ME and proENK mRNA level induced by nicotine when BAMC cells were post-treated after 9 and 12 hr. Our results suggest that long-term stimulation (for at least 6 hr) of nicotinic receptors is required for increases in proENK mRNA levels and the delayed secretion of ME in BAMC cells. The nicotine-induced delayed secretion of ME followed an increased biosynthesis of ME during the first 6 hr which appeared to result from increased transcription of the proENK gene.